Understanding of the heme biosynthetic pathway, which is essential in all organisms is currently limited by lack of structural data for the relevant enzymes. This proposal is for structure-function studies on the enzymes uroporphyrinogen decarboxylase (URO-D) and coproporphyrinogen oxidase (COPRO-OX), which catalyze successive steps in the heme biosynthetic pathway and are both associated with human diseases. Our goal is to obtain high resolution structural information by X-ray crystallography and to elucidate catalytic mechanisms by structure-based mutagenesis and biochemistry. Toward this goal we have recently determined the crystal structure of URO-D by multiwavelength anomalous diffraction at 2.0 Angstrom units resolution. Mutagenic and biochemical studies are proposed to elucidate the role of individual residues in binding and catalysis. The URO-D crystal structure reveals a dimeric arrangement that juxtaposes the deep active site clefts, and we have shown that URO-D has a 0.1 muM dissociation constant in solution. In order to test the hypothesis that URO-D dimerization is of functional importance, we also propose mutagenic and biochemical studies to determine the role of dimerization in URO-D activity. We will also determine the crystal structures of relevant URO-D mutants and inhibitor complexes. We have obtained crystals of yeast COPRO-OX and collected X-ray diffraction data to 3.0 Angstrom units resolution. This structure will also be determined and will be used to design site-directed mutants to probe the roles of specific residues. Deficiency in URO-D and COPRO-OX activities cause the disease porphyria cutanea tarda and hereditary coproporphyria, respectively. The combination of proposed structural and biochemical studies may provide the basis for the design of novel therapeutics.